1. Field of the Invention
The present invention relates to plant biotechnology. More particularly, the present invention relates a method for high efficiency protein delivery into plastids, in particular, leucoplasts.
2. The Prior Arts
Plastids are essential organelles in plants responsible for functions ranging from photosynthesis, biosynthesis of all fatty acids, starch, carotenoids, and most amino acids, to assimilation of nitrogen and sulfur. Plastids differentiate into different functional types in different tissues, for example chloroplasts in green tissues for photosynthesis, chromoplasts in petals and fruits for carotenoid pigments accumulation, and leucoplasts in non-green tissues for synthesis and storage of nutrients, including starch, proteins and oils. To perform these specific functions, different types of plastids require, and therefore import, different proteins.
Although plastids have their own genome, most plastid proteins are encoded by the nuclear genome, synthesized in the cytosol as a larger precursor with an N-terminal extension called the transit peptide. Transit peptides are necessary and sufficient for targeting passenger proteins into plastids, i.e., a transit peptide can be taken from the original precursor and fused to a passenger protein and results in delivery of the passenger protein into plastids. Transit peptides that can deliver passenger proteins into chloroplasts with high efficiency, for example the transit peptide of RuBP carboxylase small subunit precursor (prRBCS), have been identified and used to deliver passenger proteins into plastids.
Most grain- and root-type food crops, for example rice, corn and cassava, use leucoplasts to synthesize and store the starch that is used to feed the majority of the world population. However, despite the economic importance of leucoplasts, almost all of our knowledge about plastid protein import is derived from studies with chloroplasts and little is known about how proteins are imported into leucoplasts. Unfortunately, leucoplasts clearly have a different substrate preference, and transit peptides like that of prRBCS import proteins poorly into leucoplasts. For example, it has been shown that the transit peptide of prRBCS could not direct the import of the passenger protein green fluorescent protein (GFP) into leucoplasts in endosperms of transgenic wheat (Primavesi et al., 2008). Using leucoplasts isolated from castor seeds and chloroplasts isolated from pea, it has been shown that prRBCS imported much better into chloroplasts than into leucoplasts (Wan et al., 1996). Using leucoplasts and chloroplasts isolated from pea roots and leaves, respectively, it has been shown that prRBCS could not be imported into leucoplasts at all (Yan et al., 2006). Nonetheless, in these studies, no proper quantitative comparisons were performed so the import efficiency of transit peptides could not be compared directly among one another. Therefore the exact import efficiency of prRBCS transit peptide into leucoplasts is not known and no transit peptides with high leucoplast import efficiency have been discovered.
The transit peptide for prRBCS is the most widely used transit peptide for delivering engineered proteins into plastids in biotechnology applications. Examples include the famous Golden Rice, Roundup Ready® corn and Dicamba resistant soybean. As discussed above, many reports have suggested that this transit peptide deliver proteins poorly into leucoplasts and therefore is not the best transit peptide for applications that need to express proteins in leucoplasts. However, no report has performed quantitative comparisons between transit peptides. If quantitative comparison can be performed and transit peptides with higher leucoplast import efficiency than prRBCS transit peptide can be identified, these new transit peptides would be valuable tools for delivering engineered proteins into leucoplasts.